Phospholipase variants

ABSTRACT

The inventors have used protein engineering to develop variants of fungal phospholipases. Starting from a parent phospholipase, they have modified the amino acid sequence to arrive at variants which have phospholipase activity (generally, at roughly the same level as the parent enzyme) and have a lower lipase activity on triglycerides than the parent enzyme.

FIELD OF INVENTION

The present invention relates to a method of producing a polypeptide bymodifying the amino acid sequence of a polypeptide with phospholipaseactivity, to a polypeptide having phospholipase activity, and to use ofthe polypeptide in cheese-making.

BACKGROUND OF THE INVENTION

Lipolytic enzymes are polypeptides with hydrolytic activity forcarboxylic ester bonds, e.g., lipase and/or phospholipase activity. Thesubstrate specificity (relative activity on different ester bonds) isimportant for the usefulness of the lipolytic enzyme in variousindustrial applications.

WO 00/32758 discloses lipolytic enzyme variants having altered substratespecificity. WO 98/26057 discloses a Fusarium oxysporum phospholipase.WO 01/83770 describes lipase variants. WO 00/54601 describes a processfor producing cheese from cheese milk treated with a phospholipase.

SUMMARY OF THE INVENTION

The inventors have found that when a fungal phospholipase is used in acheese-making process, too high lipase activity on triglycerides maylead to a cheese product having changed properties in terms of smell andtaste, possibly due to the generation of too many free fatty acids.

To overcome this, the inventors have used protein engineering to developvariants of fungal phospholipases. Starting from a parent phospholipase,they have modified the amino acid sequence to arrive at variants whichhave phospholipase activity (generally, at roughly the same level as theparent enzyme) and have a lower lipase activity on triglycerides thanthe parent enzyme. Thus, starting from a parent fungal phospholipase (apolypeptide with phospholipase activity), the inventors have found thatthe ratio of lipase/phospholipase activity can be decreased bysubstituting a particular amino acid residue.

The variants are useful in the production of cheese, e.g. in a processor method as described in WO 00/54601, and they result in an increasedyield and at the same time avoid the changes in taste and smell, whichmay result from the generation of too many free fatty acids.

Accordingly, the invention provides a polypeptide which:

a) has phospholipase activity,

b) has an amino acid sequence which is at least 50% identical to SEQ IDNO: 1, and

c) has one or more of the following amino acids at a positioncorresponding to SEQ ID NO: 1: D62Q/E/F/W/V/P/L/G; V60R/S/K; S85Y/T;G91R/E; R125K; V203T; V228A; T231R; N233R; L259R/V/P; a deletion D266*;and/or L269A.

The invention also provides a method of producing a polypeptide,comprising:

a) selecting a first (parent) polypeptide which has phospholipaseactivity and has an amino acid sequence which is at least 50% identicalto SEQ NO: 1,

b) modifying the amino acid sequence by substituting one or more aminoacids at a position corresponding to SEQ ID NO: 1: D62Q/E/F/W/V/P/L/G;V60R/S/K; S85Y/T; G91R/E; V203T; V228A; T231R; N233R; L259R/V/P; adeletion D266*; and/or L269A, and

c) preparing a second (modified) polypeptide having the modified aminoacid sequence.

The parent polypeptide may also have lipase activity, and the method mayfurther comprise testing the lipase and phospholipase activities of thetwo polypeptides and selecting a modified polypeptide having a lowerlipase/phospholipase ratio than the parent polypeptide.

Further, the invention provides a polynucleotide encoding thepolypeptide and a method for producing cheese, comprising the steps of:

a) treating cheese milk or a fraction of the cheese milk with thepolypeptide; and

b) producing cheese from the cheese milk during or after step a).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an alignment of amino acid sequences of known fungallipolytic enzymes SEQ ID NO: 1 to 14, as follows:

1: Thermomyces lanuginosus (SWISSPROT 059952)

2: Fusarium oxysporum (U.S. Pat. No. 6,103,505 SEQ ID NO: 2, GENESEQPMW51767)

3: Absidia reflexa (U.S. Pat. No. 5,821,102 SEQ ID # 10, GENESEQPMW77403)

4: Absidia corymbifera (U.S. Pat. No. 5,821,102 SEQ ID# 6, GENESEQPMW26689)

5: Rhizomucor miehei (SWISSPROT P19515)

6: Rhizopus oryzae (SWISSPROT P21811)

7: Aspergillus niger (SWISSPROT 042807)

8: Aspergillus tubingensis (SWISSPROT 042815)

9: Fusarium heterosporum (TREMBL Q02351)

10: Aspergillus oryzae (TREMBL P78583)

11: Penicillium camemberti (SWISSPROT P25234)

12: Aspergillus foetidus (U.S. Pat. No. 5,965,422 SEQ ID # 2, GENESEQPMW33009)

13: Aspergillus niger (WO 98/31790 SEQ ID # 2, GENESEQP MW64449)

14: Aspergillus oryzae (JP 10-155493 SEQ ID # 2, GENESEQP AAW 58541)

DETAILED DESCRIPTION OF THE INVENTION

Parent Polypeptide

The polypeptide of the invention may be derived from a parentpolypeptide with phospholipase activity, particularly a phospholipaseA1, classified as EC 3.1.1.32 according to Enzyme Nomenclature(available at http://www.chem.qmw.ac.uk/iubmb/enzyme). It may be anaturally occurring fungal enzyme with phospholipase activity, e.g. oneof SEQ ID NO: 2-14, particularly a phospholipase from Fusarium oxysporumwhich is described in WO 98/26057. Alternatively, the parent may be afungal lipolytic enzyme variant with phospholipase activity as disclosedin WO 00/32758, e.g. a variant of SEQ ID NO: 1 as described in Example 5of WO 00/32758.

Lipase and Phospholipase Activities

Lipase activity is measured by the SLU method described in WO 0032758,and the lipase activity of the pure protein is expressed as SLU per unitof A280 (Absorption at 280 nm).

Phospholipase activity is measured by incubating 0.025-0.07 mg enzymeprotein (e.g. 0.05 mg) with cream (standardized to 25% fat by mixingwith skimmed milk) at 35 C for 1.5 hr without shaking and measuringphospholipid depletion (by lipid extraction and HPLC analysis).Phospholipase activity is expressed as % PL depletion.

The variant polypeptides of the invention typically show 15-75% PLdepletion by this method. The lipase activity is typically below 1000SLU/A280, particularly below 500, below 250, below 100 or below 25. ThePL/lipase ratio is typically above 0.05, particularly above 0.1, above0.2, above 0.3, above 1, above 2 or above 3.

The phospholipase activity can also be determined by known methods, e.g.as described in WO 0032758, by HPLC or by phospholipid depletion incream. Using the “monolayer phospholipase assay” described in WO0032758, the parent and the modified polypeptide may have aphospholipase activity of at least 0.25 nmol/min at enzyme dose 60 μgand 25° C.; e.g. at least 0.40 nmol/min, at least 0.75 nmol/min, atleast 1.0 nmol/min, at least 1.25 nmol/min, or at least 1.5 nmol/min.

Amino Acid Alteration

The modified polypeptide has one or more of the following amino acids ata position corresponding to the following in SEQ ID NO: 1:D62Q/E/F/W/V/P/L/G; V60R/S/K; R84G/S; S85Y/T; G91R/E; R125K; V203T;V228A; T231R; N233R; L259R/V/P; a deletion D266*; and/or L269A.Corresponding positions in SEQ ID NO: 2-14 are defined by the alignmentshown in FIG. 1, e.g. position 183 of SEQ ID NO: 2. Correspondingpositions in other sequences may be found by an alignment as describedbelow.

Compared to SEQ ID NO: 1, the polypeptide of the invention may furtherhave one or more of the following amino acids at a positioncorresponding to the following in SEQ ID NO: 1: D57G, V60G/C/K/R/L/S/Q,D62H/A, S83T, R84G/S/W; G91A/V, L93K, D96W/F/G, E99K, R125K, L259S,F262L, G263Q, L264A, I265T, G266D, T267A/E and/or L269N. Also, N- and/orC-terminus may be extended, e.g. as described in WO 9704079. Thus, theC-terminal may be extended by adding residues after position 269, e.g.addition of AGGFS or AGGFSWRRYRSAESVDKRATMTDAELEKKLNSWQMDKEWKNNQARS. TheN-terminal may br extended by the addition of amino acid residues suchas SPIRR. Such C- or N-terminal extensions should not be considered,when calculating the amino acid identity with SEQ ID NO: 1.

Sequences derived from SEQ ID NO: 2 may be C-terminal processed (e.g.during expression in A. oryzae), e.g. with positions 272, 273, 274 or286 of SEQ ID NO 2 as the C-terminal residue.

The parent and modified polypeptides may be tested for lipase andphospholipase activity, and a variant polypeptide may be selected whichhas phospholipase activity and a lipase/phospholipase ratio which islower than the parent polypeptide. Lipase activity can be determined byknown methods using a triglyceride as substrate, e.g. as described in WO00/32758.

Amino Acid Identity and Alignment

The amino acid identity may be suitably determined by means of computerprograms known in the art, such as GAP provided in the GCG programpackage (Program Manual for the Wisconsin Package, Version 8, August1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of MolecularBiology, 48, 443-45), using GAP with the following settings forpolypeptide sequence comparison: GAP creation penalty of 3.0 and GAPextension penalty of 0.1.

The variant polypeptide has an amino acid identity to SEQ ID NO: 1 whichis at least 50%, particularly at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 98%.

To find the homologous positions in lipase sequences not shown in thealignment, the sequence of interest is aligned to the sequences shown inFIG. 1. The new sequence is aligned to the present alignment in FIG. 1by using the GAP alignment to the most homologous sequence found by theGAP program. GAP is provided in the GCG program package (Program Manualfor the Wisconsin Package, Version 8, August 1994, Genetics ComputerGroup, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B.and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45).The following settings are used for polypeptide sequence comparison: GAPcreation penalty of 3.0 and GAP extension penalty of 0.1.

EXAMPLES Example 1 Construction of Variants having a IncreasedPhospholipase/Lipase Activity Ratio Compared to the Parent Enzyme

The following variant polypeptides were constructed as described in WO00/32758. Each polypeptide is described by the amino acid alterationscompared to SEQ ID NO: 1. Variant Amino acid alteration in SEQ ID NO: 11 R84W + D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N +270A + 271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 2 R84W + G91E + D96W +E99K + G263Q +L264A + I265T + G266D + T267A + L269N + 270A + 271G +272G + 273F + 274S + 275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 3V60G + D62E + R84W + G91A + D96F + E99K + G263Q + L264A + I265T +G266D + T267A + L269N 5 R84W + G91R + L93K + D96G + E99K + G263Q +L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F +274S + 275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 6 V60G + D62F +R84W + G91A + D96W + E99K + G263Q + L264A + I265T + G266D + T267A +L269N + 270A + 271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 7 R84W + S85Y + G91A +D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A +271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 8 R84W + G91A + D96W +E99K + L259V + G263Q + L264A + I265T + G266D + T267A + L269N + 270A +271G + 272G + 273F +274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 10 V60G + D62W + R84W +G91A + D96F + E99K + G263Q + L264A + I265T + G266D + T267A + L269N 11R84W + G91R + D96F + E99K + G263Q + L264A + I265T + G266D + T267A +L269N + 270A + 271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 12 V6OC + D62H + R84W +G91A + D96F + E99K + G263Q + L264A + I265T + G266D + T267A + L269N 13V60G + D62V + R84W + G91A + D96F + E99K + G263Q + L264A + I265T +G266D + T267A + L269N 14 V60K + D62L + R84W + G91A + D96F + E99K +G263Q + L264A + I265T + G266D + T267A + L269N 15 V60R + D62L + R84W +G91A + D96F + E99K + G263Q + L264A + I265T + G266D + T267A + L269N 16V60G + D62G + R84W + G91A + D96W + V228A + E99K + G263Q + L264A +I265T + G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 17 V60L + D62A + R84W +G91A + D96W + E99K + R125K + G263Q + L264A + I265T + G266D + T267A +L269N + 270A + 271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 18 D62E + R84W + G91A +D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A +271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 19 V60S + D62L + R84W +G91A + D96F + E99K + F262L + G263Q + L264A + I265T + G266D + T267A +L269N 20 D57G + V60Q + D62P + R84W + G91A + D96F + E99K + G263Q +L264A + I265T + G266D + T267A + L269N 21 R84W + G91A + D96W + E99K +L259R + G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G +272G + 273F + 274S + 275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 23D62Q + R84W + G91A + D96W + E99K + G263Q + L264A + I265T + G266D +T267A + L269N + 270A + 271G + 272G + 273F +274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 25 R84W + G91A + D96W +E99K + V203T + G263Q + L264A + I265T + G266D + T267A + L269N + 270A +271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS 26 R84S + S85T + G91A +D96S + T231R + N233R + L259P + G263Q + L264S + I265T + G266* + T267E +L269A

Each of the above variant polypeptides showed a phospholipase depletionof 15-75%, a lipase activity below 250 SLU/A280 and a PL/lipase activityabove 0.1. For comparison, a number of prior-art variants described inExample 5 of WO 0032758 were measured and were found to have a PL/lipaseratio below 0.05.

Example 2 Evaluation of Cheese Yield Using Selected Variants of theInvention

The following variant polypeptides from Example 1 were evaluated in amethod of producing cheese with the addition of a phospholipase. Thecontrols were without phospholipase addition.

The method was a bench top cheese yield evaluation test and wasperformed as described below.

1. Standardize 0.5 kg cheese milk w/ pasteurized skim milk and cream.

2. Prepare a single starter by adding 0.1 g Rhodia LH100 and 0.3 gRhodia TA061 starter cultures (for mozzarella) to 50 ml of the skim milkand equilibrate to 35° C. w/ gentle, continuous stirring.

3. Equilibrate cheese milk to 35° C. and add 0.07 mg enzyme protein perg fat, check initial pH and add 5 ml starter to each cheese milk withgentle agitation.

4. When pH reaches 6.45-6.50 add 0.5 ml of rennet (10× diluted Chymax,available from Christian Hansen); stir vigorously for three minutes thenremove stirrers from milk, cover water bath and allow milk to coagulate.

5. Cut curd at the appropriate time (30-45 minutes) wit 25 mm (½″)knives. To determine cutting time, make a downward cut into the curdwith knife or spatula. The curd is ready for cutting when the cutseparates upon lifting and sharp edges are maintained on the top surfaceat the edge of the cut. Allow the curd to rest for 5 minutes then gentlyand intermittently stir curd to prevent coalescence of curd particles.

6. Increase temperature to 41° C. and hold until curd pH reaches5.65-5.70, then drain and pour curd particles into stainless steelbowls. Float bowls in 41° C. water bath to maintain curd temperature.Periodically drain excess whey, leaving only enough to cover curds formaintenance of heat.

7. When curd pH ˜5.25-5.3, drain all whey and flood curd w/ D.I. waterat 57° C. for 5 min. Stretch the curd by hand for ˜1 min in 59° C.water, then place the curd in ice water for 15 min and dry blot. Recordweight of curd and refrigerate until further analysis.

Results

Variants No. 2, 4, 5, 8, 9, 10, 16, 22 and 24 of Example 1 were tested.All the tested variants resulted in improved yield compared to thecontrol, when calculated as moisture adjusted yield.

1-7. (canceled)
 8. A polypeptide which: a) has phospholipase activity,a) has an amino acid sequence which is at least 50% identical to SEQ IDNO: 1, and b) has one or more of the following amino acid alterations:D62Q/E/F/W/V/P/L/G; V60R/S/K; S85Y/T; G91R/E; R125K; V203T; V228A;T231R; N233R; L259R/V/P; a deletion D266*; and/or L269A (using SEQ IDNO:1 for numbering).
 9. The polypeptide of claim 8, which has one ormore of the following amino acids alterations D57G, V60G/C/L/Q, D62H/A,S83T, R84G/S/W; G91A/V, L93K, D96W/F/G, E99K, R125K, L259S, F262L,G263Q, L264A, I265T, G266D, T267A/E, L269N and/or by a C-terminalextension.
 10. The polypeptide of claim 9, wherein the C-terminalextension is AGGFS or AGGFSWRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS.11. The polypeptide of claim 8, which has the sequence of SEQ ID NO: 1with one of the following sets of alterations: R84W + D96W + E99K +G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G +273F + 274S + 275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS R84W +G91E + D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N +270A + 271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS V60G + D62E + R84W +G91A + D96F + E99K + G263Q + L264A + I265T + G266D + T267A + L269NR84W + G91R + L93K + D96G + E99K + G263Q + L264A + I265T + G266D +T267A + L269N + 270A + 271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS V60G + D62F + R84W +G91A + D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N +270A + 271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS R84W + S85Y + G91A +D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A +271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS R84W + G91A + D96W +E99K + L259V + G263Q + L264A + I265T + G266D + T267A + L269N + 270A +271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS V60G + D62W + R84W +G91A + D96F + E99K + G263Q + L264A + I265T + G266D + T267A + L269NR84W + G91R + D96F + E99K + G263Q + L264A + I265T + G266D + T267A +L269N + 270A + 271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS V6OC + D62H + R84W +G91A + D96F + E99K + G263Q + L264A + I265T + G266D + T267A + L269NV60G + D62V + R84W + G91A + D96F + E99K + G263Q + L264A + I265T +G266D + T267A + L269N V60K + D62L + R84W + G91A + D96F + E99K + G263Q +L264A + I265T + G266D + T267A + L269N V60R + D62L + R84W + G91A + D96F +E99K + G263Q + L264A + I265T + G266D + T267A + L269N V60G + D62G +R84W + G91A + D96W + V228A + E99K + G263Q + L264A + I265T + G266D +T267A + L269N + 270A + 271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS V60L + D62A + R84W +G91A + D96W + E99K + R125K + G263Q + L264A + I265T + G266D + T267A +L269N + 270A + 271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS D62E + R84W + G91A +D96W + E99K + G263Q + L264A + I265T + G266D + T267A + L269N + 270A +271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS V60S + D62L + R84W +G91A + D96F + E99K + F262L + G263Q + L264A + I265T + G266D + T267A +L269N D57G + V60Q + D62P + R84W + G91A + D96F + E99K + G263Q + L264A +I265T + G266D + T267A + L269N R84W + G91A + D96W + E99K + L259R +G263Q + L264A + I265T + G266D + T267A + L269N + 270A + 271G + 272G +273F + 274S + 275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS D62Q +R84W + G91A + D96W + E99K + G263Q + L264A + I265T + G266D + T267A +L269N + 270A + 271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS R84W + G91A + D96W +E99K + V203T + G263Q + L264A + I265T + G266D + T267A + L269N + 270A +271G + 272G + 273F + 274S +275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS R84S + S85T + G91A +D96S + T231R + N233R + L259P + G263Q + L264S + I265T + G266* + T267E +L269A


12. The polypeptide or claim 8, which has an amino acid sequence whichis at least 60% identical to SEQ ID NO:
 1. 13. The polypeptide or claim8, which has an amino acid sequence which is at least 70% identical toSEQ ID NO:
 1. 14. The polypeptide or claim 8, which has an amino acidsequence which is at least 80% identical to SEQ ID NO:
 1. 15. Thepolypeptide or claim 8, which has an amino acid sequence which is atleast 90% identical to SEQ ID NO:
 1. 16. The polypeptide or claim 8,which has an amino acid sequence which is at least 95% identical to SEQID NO:
 1. 17. The polypeptide or claim 8, which has an amino acidsequence which is at least 98% identical to SEQ ID NO:
 1. 18. Apolynucleotide encoding the polypeptide of claim
 8. 19. A method ofproducing a polypeptide, comprising: a) selecting a first polypeptidewhich has phospholipase activity and has an amino acid sequence which isat least 50% identical to SEQ NO: 1, b) altering the amino acid sequencewherein the alteration comprises one or more substitutions or deletioncorresponding to the following in SEQ ID NO: 1: D62Q/E/F/W/V/P/L/G;V60R/S/K; S85Y/T; G91 R/E; V203T; V228A; T231R; N233R; L259R/V/P; adeletion D266*; and/or L269A.
 20. A method for producing cheese,comprising the steps of: a) treating cheese milk or a fraction of thecheese milk with the polypeptide claim 8; and b) producing cheese fromthe cheese milk during or after step a).